The genetic material of all known living organisms is deoxyribonucleic acid (DNA), except in certain viruses whose genetic material may be ribonucleic acid (RNA). DNA consists of a chain of individual deoxynucleotides chemically linked in specific sequences. Each deoxynucleotide contains one of the four nitrogenous bases which may be adenine (A), cytosine (C), guanine (G) or thymine (T), and a deoxyribose, which is a pentose, with a hydroxyl group attached to its 3' position and a phosphate group attached to its 5' position. The contiguous deoxynucleotides that form the DNA chain are connected to each other by a phosphodiester bond linking the 5' position of one pentose ring to the 3' position of the next pentose ring in such a manner that the beginning of the DNA molecule always has a phosphate group attached to the 5' carbon of a deoxyribose. The end of the DNA molecule always has an OH (hydroxyl) group on the 3' carbon of a deoxyribose.
DNA usually exists as a double-stranded molecule in which two antiparallel DNA strands are held together by hydrogen bonds between the bases of the individual nucleotides of the two DNA strands in a strictly matched "A--T" and "C--G" pairing manner. It is the order or sequence of the bases in a strand of DNA that determines a gene which in turn determines the type of protein to be synthesized. Therefore, the accurate determination of the sequence of the bases in a DNA strand which also constitutes the genetic code for a protein is of fundamental importance in understanding the characteristics of the protein concerned.
The process used to determine the sequence of the bases in a DNA molecule is referred to as DNA sequencing. Among the techniques of DNA sequencing, the enzymatic method developed by Sanger et al. (1) is most popular. It is based on the ability of a DNA polymerase to extend a primer annealed to the DNA template to be sequenced in the presence of four normal deoxynucleotide triphosphates (dNTPs), namely, DATP, dCTP, dGTP and dTTP, and on the ability of the nucleotide analogs, the dideoxynucleotide triphosphates (ddNTPs), namely, ddATP, ddCTP, ddGTP and ddTTP, to terminate the extension of the elongating deoxynucleotide polymers at various lengths.
In the classic one-step Sanger method, the sequence determination is carried out in a set of four separate tubes, each containing all four normal dNTPs, one of which is labeled with a radioactive isotope, .sup.32 P or .sup.35 S, for autoradiographic localization, a limiting amount of one of the four ddNTPs, a DNA polymerase, a primer, and the DNA template to be sequenced. As a result of the DNA polymerase activity, individual nucleotides or nucleotide analogs are added to the new DNA chains, all starting from the 3' end of the primer in a 5'-3' direction, and each linked to adjacent ones with a phosphodiester bond in a base sequence complementary to the DNA sequence of the template. Inasmuch as there is a nucleotide analog in the reaction mixture, each tube eventually contains numerous newly formed DNA strands of various lengths, all ending in a particular ddNTP, referred to as A, C, G or T terminator.
After resolving the four sets of reaction products by high-resolution polyacrylamide/urea gel electrophoresis, the populations of the newly formed DNA strands are separated and grouped according to their molecular weight. An autoradiographic image of the gel will show the relative positions of these DNA strands as bands which differ from one another in distance measured by one nucleotide in length, all sharing an identical primer and terminating with a particular ddNTP (A, C, G or T). By reading the relative positions of these bands in the "ladder" of the autoradiograph, the DNA sequence of the template can be deduced.
The DNA polymerase used in the reaction mixture plays a pivotal role in DNA sequencing analysis. To be useful for DNA sequencing, a DNA polymerase must possess certain essential properties. For example, it must have its natural 5'-3' exonuclease activity removed by mutagenesis or by posttranslational modification, such as enzymatic digestion, and must be able to incorporate dNTPs and ddNTPs, without undue discrimination against ddNTP and with a sufficiently high processivity which refers to the ability of the enzyme to polymerize nucleotides onto a DNA chain continuously without being dislodged from the chain, and a sufficiently high elongation rate. A 5'-3' exonuclease activity associated with a DNA polymerase will remove nucleotides from the primer, thus cause a heterogeneous 5' end for the newly formed DNA strands, resulting in a false reading of the strand lengths on the sequencing gel. A DNA polymerase with a low processivity and a low elongation rate will cause many undesirable noise background bands of radioactivity due to the presence of DNA strands which are formed with improper lengths and improper terminations. Among the more commonly used DNA polymerases, Sequenase.TM. has a higher processivity and a higher elongation rate than others, such as the Klenow fragment, Taq, and Vent polymerases (2), and is therefore one of the most popular DNA polymerase selected for DNA sequencing to-date.
However, even when a DNA polymerase has been endowed with all the essential properties listed above, it may still generate erroneous or misleading band patterns of radioactivity in the sequencing gel. These artifactual patterns do not faithfully reflect the true nucleotide sequence in the template being sequenced. They may be caused by premature termination of the elongating strands due to the presence of secondary structures formed along the template, such as "hairpins" in the regions that contain palindromic sequences or that are rich in G and C bases (3); or, they may occur as a result of inadequate "proof-reading" function of the DNA polymerase that will allow the removal of misincorporated nucleotides at the 3' end of an elongating strand.
Researchers in the field of DNA sequencing often have to use several approaches to confirm their findings in order to avoid being misled by these potentially erroneous sequence data. For example, they sometimes rely on repeating the same sequencing experiment with different DNA polymerases, or performing another sequencing reaction with the template which is complementary to the first single-stranded DNA template, and compare the results for possible discrepancies.
Numerous investigators have tried to find an ideal DNA polymerase for enzymatic sequencing, i.e. an enzyme that not only has all the essential properties required for sequencing reaction, but also is capable of resolving the secondary hairpin structures and preventing the formation of strands containing nucleotides non-complementary to those of the template being sequenced.
The discovery by Ye and Hong (4) of the thermostable large fragment of DNA polymerase isolated from Bacillus stearothermophilus (Bst), an enzyme that is functional over the temperature range between 25.degree. C. and 75.degree. C., but is most active at 65.degree. C., and possesses all the essential properties for DNA sequencing, has largely solved the problem caused by secondary structures in the template since these secondary structures are destabilized when the sequencing reaction is carried out at 65.degree. C. In the past few years since this enzyme was made commercially available under the name of Bst DNA Polymerase (Bio-Rad Laboratories), independent reports have confirmed that during sequencing reaction catalyzed by this enzyme all four dNTPs, including dCTP, and other nucleotide analogs, such as dITP and 7-deaza-dGTP, are incorporated equally effectively in the chain elongation, thus eliminating the weak "C" band phenomena often observed when other DNA polymerases are used, and producing a very good band uniformity on the sequencing gel. It has been further established that at this elevated temperature Bst DNA polymerase system can be used both for the classic Sanger one-step reaction as well as for the "labeling/termination" sequencing reaction, double-stranded DNA sequencing, and the incorporation of .sup.35 S-labeled nucleotides, and .sup.32 P-labeled nucleotides. Since this system can be placed at room temperature for at least two weeks without significant loss of its enzymatic activity, it has been adapted for automation of DNA sequencing which requires a stable DNA polymerase, using either fluorescent dye or radioactive isotope labeling. (See also 9, 12, and 13.)
However, when this Bst enzyme is used for automated fluorescent DNA sequencing, only partially satisfactory results have been obtained with fluorescent dye-labeled primers (see 12 and EG Bulletin 1771 of Bio-Rad Laboratories), and even less satisfactory results are obtained with fluorescent dye-labeled ddNTP terminators. Even when fluorescent dye-labeled primers are used, a significant number of mismatched ddNTPs are incorporated onto the 3' end of the extending nucleotides in the enzymatic reaction, thus generating erroneous sequencing data (see Bio-Rad EG Bulletin 1771). With this in mind, the inventors sought, and found, a better DNA polymerase for DNA sequencing, especially for automated fluorescent dye-labeled primer and fluorescent dye-labeled terminator sequencing.
Another disadvantage of the Bst DNA polymerase currently known in the art is its lack of 3'-5' exonuclease activity (5), and specifically, proof-reading 3'-5' exonuclease activity. A survey of the sequencing data collected from fourteen research centers which have used this Bst DNA polymerase for their DNA sequencing work on over 120 DNA clones showed that, statistically, base pair mismatching occurs at a rate of about 1.5.times.10.sup.-5. That is, approximately 1.5 errors can be expected in one hundred thousand nucleotide incorporations during nucleotide polymerization catalyzed by the enzyme.
It is generally known that the formation of incorrect DNA sequences due to mismatching of base pairs between the template and the growing nucleotide chain in DNA sequencing may be prevented by a 3'-5' exonuclease activity which "proof-reads" the nucleotide chain. However, even if a DNA polymerase exhibits 3'-5' exonuclease activity in vitro, it is often the case that the polymerase will not adequately "proof-read". Thus, the polymerase will not be capable of removing mismatched nucleotides from a newly formed DNA strand as efficiently as those nucleotides correctly matched with the nucleotides of the template. In other words, a 3'-5' exonuclease may excise the correctly matched nucleotides at a faster rate than the mismatched ones from the 3' terminus, or excise both the correctly matched and the mismatched nucleotides at the same rate. Consequently, even where the DNA polymerase has 3'-5' exonuclease activity, it does not perform any useful proof-reading function during DNA polymerization.
It is also known that a 3'-5' exonuclease activity associated with a DNA polymerase, in the presence of low concentrations of dNTPs, often counteracts the normal chain elongation process catalyzed by the polymerase, induces cyclic incorporation and degradation of nucleotides over the same segment of template, or even operates more efficiently than the polymerase activity per se, to the extent of causing degradation of the primer. Consequently, removal of the 3'-5' exonuclease activity along with the 5'-3' exonuclease activity from the native DNA polymerases by chemical means or by genetic engineering techniques has become a standard procedure in producing DNA polymerases for sequencing. This is a common strategy to preserve the essential properties of a DNA polymerase.
For example, among the major commercially available sequencing enzymes (other than the native Taq (Thermus aquaticus) DNA polymerase which lacks a 3'-5' exonuclease activity de novo) the 3'-5' exonuclease activity has been removed from the native T7 DNA polymerase, which lacks a 5'-3' exonuclease, either by a chemical reaction that oxidizes the amino acid residues essential for the exonuclease activity (Sequenase.TM. Version 1) or genetically by deleting 28 amino acids essential for the 3'-5' exonuclease activity (Sequenase.TM. 2).
Vent.sub.R (exo.sup.-) DNA polymerase, which is recommended as the preferred form of the Vent DNA polymerase for sequencing, also has its 3'-5' exonuclease activity removed by genetic modification. The native Vent DNA polymerase and the Klenow fragment isolated from the native E. coli DNA polymerase I possess a 3'-5' exonuclease; but these enzymes are no longer considered the enzymes of choice for DNA sequencing.
The currently known Bst DNA polymerase (e.g., produced by Bio-Rad Laboratories) isolated and purified from the cells of Bacillus stearothermophilus for DNA sequencing is free of 3'-5' exonuclease activity (5).
IsoTherm.TM. DNA Polymerase, a commercially available Bst DNA polymerase for DNA sequencing, marketed by Epicentre Technologies (1402 Emil Street, Madison, Wis. 53713), is also based on a Bst DNA polymerase whose 3'-5' exonuclease activity has been enzymatically removed (6).
Only the rBst DNA Polymerase produced from an over-expressing recombinant clone in E. coli, which is the product of the DNA pol I gene of Bacillus stearothermophilus, possesses a 3'-5' exonuclease activity in addition to a 5'-3' exonuclease activity. However, due to the existence of an undesirable 5'-3' exonuclease activity and a 3'-5' exonuclease activity of unknown characteristics, the latter product is not recommended by the company for DNA sequencing (6).
Over the past 10 years there has been a trend to develop and improve the automated fluorescent DNA sequencing technology to replace the classic radioactive isotope labeling manual method for DNA sequencing because of the potential harmful effects of the radioactive materials to humans and because of the need for automated high throughput DNA sequencing systems. In using fluorescent dyes as markers for labeling the DNA strands generated in enzymatic reactions for sequencing, the dyes can be either coupled with the primer, or coupled with the ddNTP terminators, namely the dye-labeled ddATP, dye-labeled ddCTP, dye-labeled ddGTP and dye-labeled ddTTP. Sequencing techniques based on these two forms of labeling of the final enzymatic reaction products are commonly referred to as "dye primer sequencing" and "dye terminator sequencing", respectively.
In the dye primer sequencing, ddNTPs are employed as the chain terminators, as in the original classic Sanger method which uses radioactive isotope as the marker. The molecular structure of ddNTPs are almost identical to that of dNTPs, the natural building blocks of all DNA molecules. Therefore, any DNA polymerase which has been used for radioactive isotope manual DNA sequencing can be easily adapted for fluorescent dye primer DNA sequencing with equally satisfactory results. The disadvantage in the dye primer technology is that the primer for each template to be sequenced must be labeled with four different fluorescent dyes and that the enzymatic reaction must be performed in four separate test tubes each containing only one of the ddNTPs, namely ddATP, ddCTP, ddGTP or ddTTP, as in the classic Sanger radioisotope method.
In the dye terminator technology for DNA sequencing, the fluorescent dye-labeled ddATP, dye-labeled ddCTP, dye-labeled ddGTP and dye-labeled ddTTP are coupled with different fluorescent dyes, each emitting a specific light spectrum, thus directly reporting the type of ddNTP at the 3' terminus of the DNA fragment. Unlike the situations in the dye primer technology in which four different fluorescent dyes are coupled to a primer incorporated into all newly formed DNA strands, these dye-labeled ddNTPs serve the dual function of a specific base terminator and a "color marker". There is no need to label the primer for each new template, and the polymerase DNA extension reaction can be performed in a single test tube to generate the required specifically terminated and specifically dye-labeled DNA fragments of various sizes for DNA sequencing.
The advantage of using fluorescent dye-labeled terminators for DNA sequencing is obvious. However, there are certain difficulties to overcome before an enzymatic reaction system suitable for a radioisotope technique or suitable for a dye primer technique can be adapted for a dye terminator technology. An increase of the molecular weight from less than 500 for a ddNTP terminator to about 800 or more for a fluorescent dye-labeled ddNTP terminator may be associated with potential three-dimensional structural changes. These molecular alterations may interfere with the process of incorporation of the dye-labeled ddNTPs as chain terminators by the DNA polymerase to the 3' end of an extending DNA strand in terms of lowering the rate of incorporation, lowering the processivity of the enzyme for this new substrate, reducing the enzyme-terminator binding specificity and changing the enzyme-terminator binding kinetics.
For example, both Taq DNA polymerase and Sequenase II.TM. (a T7 DNA polymerase) have been used for radioisotope labeling DNA sequencing with excellent results, and have been adapted for fluorescent dye-labeled primer DNA sequencing. But neither can be used for fluorescent dye-labeled terminator DNA sequencing technologies. As reported in U.S. Pat. No. 5,614,365, when the Taq DNA polymerase was used for fluorescent dye-labeled terminator chemical reactions, the reaction products generated no readable data on the DNA sequencer. Most of the fluorescence was either in unincorporated dye-ddNTPs at the leading front of the test gel, or in fragments greater than several hundred bases in length. Using a Taq DNA polymerase mutant in which the amino acid, phenylalanine, at position 667 of its amino acid sequence has been replaced by a tyrosine and which has an increased ability to incorporate dideoxynucleotides (6,000 times more efficient), to replace the unmodified Taq DNA polymerase for the experiment, the results are significantly improved. This F667Y mutant of Taq DNA polymerase is now marketed by Amersham Life Science, Inc. under the trademark ThermoSequenase.TM.. It is used for cycle-sequencing in which the enzymatic reaction mixture is subjected to numerous cycles of extension-termination, denaturing and annealing to ensure that sufficient dye-terminator-labeled enzymatic reaction products are generated for the DNA sequencing procedure. Because of the low processivity of the parent Taq DNA polymerase, ThermoSequenase.TM. is not recommended for direct DNA sequencing without precyclings. Like Taq DNA polymerase, ThermoSequenase T lacks a proof-reading exonuclease activity.
Bacillus stearothermophilus, Bacillus caldotenax and Bacillus caldolyticus are classified as mesophilic microbes; although their DNA polymerases are referred to as thermostable (most active at 65.degree. C.) they are inactivated at 70.degree. C. or above. This is contrasted with other enzymes, such as Taq, which are truly thermophilic--that is, their DNA polymerases tolerate and remain active at temperatures higher than 95.degree. C. These mesophilic bacillus strains, especially Bacillus stearothermophilus, produce DNA polymerases that are useful in DNA sequencing applications. However, a disadvantage of the DNA polymerases of these strains is that during DNA sequencing they all exhibit a high degree of selective discrimination against incorporation of certain particular members of fluorescent dye-labeled ddNTPs, namely the fluorescent dye-labeled ddCTP and fluorescent dye-labeled ddATP, as terminators onto the 3' end of the extending DNA fragments during enzymatic reaction. This peculiar characteristic of selective discrimination against incorporation of fluorescent dye-labeled ddCTP and ddATP of the natural DNA polymerases isolated from Bacillus stearothermophilus and Bacillus caldotenax was not previously recognized. Such selective discrimination is apparently sequence-related, and cannot be corrected or compensated by mere adjustment of the concentrations of the dNTPs.
Thus, there is a need for a mesophilic bacillus DNA polymerase that does not selectively discriminate against incorporation of fluorescent dye-labeled ddCTP and ddATP, during dye terminator DNA sequencing.